Characterization of the ATF/CREB site and its complex with GCN4.
We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations. Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor. The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove. Monte Carlo simulations quantitate the ATF/CREB site structure as an 8 degrees bend toward the major groove in a coordinate frame near the center of the site. The ATF/CREB site is underwound by 53 degrees relative to the related AP-1 site DNA. The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1 degrees) in intrinsic bend angle. Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.
Hockings
S C
SC
Department of Chemistry, 225 Prospect Street, Yale University, New Haven, CT 06511, USA.
Kahn
J D
JD
Crothers
D M
DM
D001665:Binding Sites; D002474:Cell-Free System; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005656:Fungal Proteins / Q000737:chemistry*; D007700:Kinetics; D009010:Monte Carlo Method; D009038:Motion; D009690:Nucleic Acid Conformation; D011494:Protein Kinases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
D017362:Cyclic AMP Response Element-Binding Protein; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases